A complete deletion of one copy of the PIN1 gene rescues pid null mutants.

A) PIN1 gene is deleted using CRISPR/Cas9 gene editing technology. The two guide RNA target sites are shown with PAM underlined and in bold. The PIN1 start codon ATG is highlighted in yellow. The sequences flanking the deletions are shown. The two mutants have less than 18 bp of PIN1 coding sequence and are unlikely to produce any PIN1 proteins. B) Heterozygous pin1 full #70, which had a deletion of 3078 bp of the PIN1 gene, restored the fertility of pid-TD1, a T-DNA insertion mutant that is completely sterile in WT PIN1 background. C) pin1 full #32, when heterozygous, rescued pid-c1, a null pid allele.

Overexpression of NPY1 rescues pid null mutants and triggers phosphorylation of PIN proteins.

A) The NPY1 overexpression (OE) line #68 rescues the pid-c1, a null allele. Note that pid-c1 makes pin-like inflorescences and is completely sterile. pid-c1 no longer makes pin-like structures and is completely fertile when NPY1 protein levels are increased. A second NPY1 overexpression line (#83) also restored the fertility of pid-c1 (Supplemental Figure S2). B) NPY1 protein levels in the NPY1 OE lines in WT background and in pid mutants. In line #68, NPY1 protein level is 8.0-fold higher than WT while line #83 has 9.7-fold higher NPY1 protein level than WT. In the absence of PID, overexpression of NPY1 leads to slightly lower NPY1 protein levels than the same transgenic event in WT background. C) Overexpression of NPY1 leads to an increase of phospho-peptides. The volcano plot shows the fold changes (Log2 scale) of phospho-peptide levels in NPY1 OE lines over WT. Data above the horizonal dotted line are statistically significant. The vertical dotted line marks 2-fold change. D) Overexpression of NPY1 leads to phosphorylation of PIN proteins. Among the phospho-peptides that displayed the most differences between NPY1 OE lines and WT are peptides from NPY1 and PIN proteins. In fact, in the right up-Quadrant in C, the only peptides were from NPY1 and PIN proteins.

Overexpression of NPY1 leads to increases of phosphorylation of PIN proteins.

NPY1 in WT 68 refers to NPY1 overexpression (OE) line #68 in WT background while WT68 refers to plants without the NPY1 OE construct segregated from the NPY1 OE line #68. NPY1 in pid68 refers to the NPY1 OE line #68 in the pid-c1 homozygous background. The comparisons were made on basis of the same transgenic event of integrating the NPY1 OE construct into Arabidopsis genome. Similar nomenclature is used for line #83. Fold change over 1.5 is highlighted in orange and P-value less than 0.05 is highlighted in green.

Overexpression of the truncated NPY1 lacking the C-terminal 30 amino acid residues (NPY1ΔC) leads to smaller plants, agravitropic roots, and auxin resistance.

A) Overexpression of NPY1ΔC leads to small plant stature. The NPY1ΔC plants (two independent lines, #35 and #4) have short petiole and epinastic leaves. B) NPY1ΔC plants have smaller flowers and are slower in developing siliques. C) NPY1ΔC #35 is much shorter than WT. NPY1ΔC plants often have small pin-like structures (arrow). D) A pin-like structure (arrow) of NPY1ΔC #35 plants. E) NPY1ΔC plants have lost normal gravitropic responses. F) NPY1ΔC plants are auxin-resistant. Top panel, 5-day old seedlings were transferred to MS media containing 100 nM 2,4-Dichlorophenoxyacetic acid (2,4-D) and the root tips were marked. Bottom panel: The same plants from the top panel have grown for three more days. Note that roots of WT plants stopped growing, while roots of NPY1ΔC plants continued to grow. 2,4-D did not rescue the defects of gravitropic responses. Plants from left to right: two NPY1ΔC #4, two WT, two WT, two NPY1ΔC #35.

Overexpression of the truncated NPY1 lacking the C-terminal 30 amino acid residues (NPY1ΔC) increases phosphorylation of PIN2 and other PINs.

A) NPY1ΔC levels are up several folds in two independent NPY1ΔC overexpression lines (Line 4 and Line 35). B) Overexpression of NPY1ΔC led to increases of phosphorylation of many proteins. The phospho-peptides that were at least 2-fold enriched in NPY1ΔC OE lines compared to WT and that are statistically significant are shown in the up-right-Quadrant. C) Among the top 50 enriched phospho-peptides in NPY1 ΔC OE lines compared to WT, many are from NPY1 (blue dots) and PIN proteins. D) PIN2 phosphorylation increased by at least 2-fold in both line 4 and line 35 for the three detected PIN2 peptides. Peptide I is SESGGGGsGGGVGVGGQNK, peptide II refers to KGsDVEDGGPGPR, and peptide III is HGYTNsYGGAGAGPGGDVYSLQSSK.

The flower initiation pathway and phototropic pathway use analogous signaling mechanisms.

A) Plasma-membrane-localized phototropins perceive blue light, causing changes in phosphorylation status of NPH3 and NPH3-PHOT association. In the nucleus, transcription factor ARF7/NPH4 also plays a role. B) Pathway for auxin-mediated flower initiation. Three genes (PID, NPY, and MP) are homologous to their counterparts in phototropism (color coded). The dotted arrows indicate that there are gaps in our understanding of the step. PHOT: PHOTOTROPIN; NPH3: NON-PHOTOTROPICAL HYPOCOTYL 3; ARF7: AUXIN RESPONSE FACTOR 7; MP: MONOPTEROS.